Project Summary In the proposed work, I will determine the role of the Tribbles homologue 1 (Trib1) in the development and function of granulocytes, specifically eosinophils and neutrophils. These cells are critical to prevent infection but can mediate immunopathology. Our lab and others identified Trib1 as a regulator of myeloid development. Trib1-deficient mice lack eosinophils and concomitantly expand neutrophils. This correlated with increased proteins levels of the myeloid transcription factor, C/EBP?. Despite this, it is unknown when developmentally Trib1 regulates granulopoiesis and how Trib1 controls progenitor plasticity. I observe that Trib1-deficient mice develop an atypical population of bone marrow granulocytes with both eosinophilic and neutrophilic features, suggestive of altered fate regulation. I hypothesize that Trib1 controls granulocyte identity as cells differentiate from lineage-committed progenitors. Critically, it is unknown if Trib1 can modulate mature granulocyte function in addition to development. I observe that Trib1-deficient neutrophils produce more of the pro-inflammatory cytokine TNF-? with LPS stimulation. I hypothesize that this due to a newly identified interaction between Trib1 and the NF-?B regulator MALT1, a pathway known to control inflammatory function. Despite this, it is unknown how Trib1 controls activation and if the Trib1:MALT1 interaction regulates cell function. In Aim 1, I will investigate the role of Trib1 in regulating cell fate plasticity. I will localize the developmental stage of Trib1-mediated regulation using ex vivo cultures of myeloid progenitors with inducible Trib1 deficiency. To investigate fate plasticity in vivo, I will use a fluorescent reporter to track the fate of cells that simultaneously commit to the eosinophil lineage and delete Trib1, hypothesizing that the mature neutrophil pool will contain cells derived from eosinophil progenitors. In addition, I will evaluate how C/EBP? contributes to Trib1-mediated regulation of granulopoiesis. In Aim 2, I will evaluate a novel role of Trib1 in regulating mature neutrophil activation. I will determine how Trib1-deficient neutrophils respond to inflammatory stimuli with the hypothesis that they are hyper-active. As I hypothesize that Trib1 interacts with MALT1 and suppresses NF-?B activation, I will measure NF-?B pathway activation following stimulation, using a genetic inhibitor of NF-?B to inhibit activation in Trib1-deficient cells. I will subsequently perform structure/function analysis to map the Trib1:MALT1 interaction. Finally, I will assess the function of this interaction by expressing a Trib1-mutant unable to bind MALT1 in Trib1-deficient neutrophils. This work will provide critical insights into the regulation of granulocyte development and identify novel targets for therapy in neutrophil-mediated inflammation.